It has been demonstrated by the applicant that demineralized bone matrix induces new bone formation when inplanted in soft tissue. Freshly excised bone, demineralized in cold dilute HCl for 48 hours and implanted in brain, muscle subcutis, and bone defects, induces differentiation of postfetal mesenchymal-type perivascular connective tissue cells into cartilage and bone (Urist, M. R., Science, 150: 893-899, 1965). The process is generally designated as matrix induced bone formation. Further, the inductive principle has been discovered and extracted; it is a material designated bone morphogenetic protein (BMP). Reference is made to U.S. Pat. Nos. 4,294,753; 4,455,256; and application Ser. No. 523,606, filed Aug. 16, 1983 and now U.S. Pat. No. 4,619,989. See also: Urist, M. R., et al., Proc. Soc. Exp. Biol. Med., 173: 194-199, 1983; and, Urist, M.R., et al., Proc. Natl. Acad. Sci. (USA) 81: 371-375, 1984. These patents, application and references are incorporated herein by reference.
It is believed that BMP transforms tissue cells into osteoblasts, cells that manufacture bone. Hypothetically, the process is classified as phenotypic transformation and the BMP is a paracrine molecule. As distinguished from malignant transformation by a carcinogen, phenotypic transformation is a self-limited host-regulated development process. During a process that replicates normal human fetal development, BMP-induced osteoblasts to form cartilage, which, over a period of several months, evolved into solid bone. The use of BMP implants by physicians offers considerable advantages over traditional bone graft operations. The list of present and potential uses for BMP includes almost every site in the human body: for use in replacing bone that has been destroyed by disease or accident; for use in treatment of scoliosis victims; for use in treatment of mal or misformed bone; for use in healing two edges of a fracture, and the like.
Currently, the process for isolation of BMP is relatively lengthy and expensive. Whereas milligram doses of BMP are required to induce bone formation in vivo, only microgram and picogram doses of hormones, vitamins and metabolites are needed to produce measureable biological reactions. One objective of the present invention was to identify and produce functional peptide segments of BMP that yield enhanced biological activity per unit of implanted bone morphogenetic material. Another objective was to create and produce biologically active peptides that are sequenced and then produced in quantity by direct biochemical synthesis from the constituent amino acids, for example, by the Merrifield method. Another objective of the present invention was to provide bone morphogenetic peptides that are sequenced and produced in quantity by expression of "genes" by recombinant DNA technology, or are used to construct nucleic acid screening probes for the isolation of the gene comprising the osteoinductive region of BMP for expression of bone morphogenetic peptides by recombinant DNA technology. Obtaining and characterizing peptide segments with bone morphogenetic activity is a key step in several commercially feasible processes to manufacture in quantity an efficient bone morphogenetic agent.